Methods, reagents and kits for flow cytometric immunophenotyping

Abstract

The invention relates to the field of flow cytometry and more particularly to a panel of antibody reagents conjugated to fluorescent compounds. Provided are reagent compositions, comprising at least eight distinct fluorochrome-conjugated antibodies comprising a set of at least there identification antibodies for the identification of a leukocyte population of interest and at least four characterization antibodies for further characterization and/or classification of said leukocyte population. Also provided are kits and methods related to the reagent compositions.

The invention claimed is: 
     
       1. A reagent composition for flow cytometric immunophenotyping of leukocytes and characterization of normal and aberrant subpopulations thereof, wherein the reagent composition comprises fluorochrome-conjugated antibodies directed against the following combination of markers:
 CD20, CD4, CD45, CD19, Igλ, CD8, Igκ, CD56, CD5, T-cell receptor (TCR) γδ, CD3, and CD38, wherein:
 (i) the antibody against CD20 and the antibody against CD4 are both conjugated to the same fluorochrome; 
 (ii) the antibody against Igλ and the antibody against CD8 are both conjugated to the same fluorochrome; 
 (iii) the antibody against CD19 and the antibody against TCRγδ are both conjugated to the same fluorochrome, and 
 (iv) the antibody against Igκ and the antibody against CD56 are both conjugated to the same fluorochrome, and 
 wherein: 
 the antibody against CD45, the antibody against CD5, the antibody against CD3, and the antibody against CD38 are each conjugated to a different fluorochrome; and 
 the fluorochrome in (i), the fluorochrome in (ii), the fluorochrome in (iii), the fluorochrome in (iv), the fluorochrome attached to the antibody against CD45, the fluorochrome attached to the antibody against CD5, the fluorochrome attached to the antibody against CD3, and the fluorochrome attached to the antibody against CD38, each having a distinguishably distinct fluorescence emission. 
 
 
     
     
       2. A set of at least two reagent compositions, said set comprising the reagent composition according to  claim 1  and at least one further reagent composition comprising distinguishably distinct fluorochrome-conjugated antibodies directed against one of the following combinations of markers:
 (i) CD20, CD45, CD23, CD10, CD79b, CD19, CD200, and CD43, wherein the antibody against CD20, the antibody against CD45, the antibody against CD23, the antibody against CD10, the antibody against CD79b, the antibody against CD19, the antibody against CD200, and the antibody against CD43 are each conjugated to a different fluorochrome having a distinguishably distinct fluorescence emission; 
 (ii) CD20, CD45, CD31, leukocyte associated immunoglobulin like receptor 1 (LAIR), CD11c, CD19, IgM, and CD81, wherein the antibody against CD20, the antibody against CD45, the antibody against CD31, the antibody against LAIR1, the antibody against CD11c, the antibody against CD19, the antibody against IgM, and the antibody against CD81 are each conjugated to a different fluorochrome having a distinguishably distinct fluorescence emission; 
 (iii) CD20, CD45, CD103, CD95, CD22, CD19, CXCR5, and CD49d, wherein the antibody against CD20, the antibody against CD45, the antibody against CD103, the antibody against CD95, the antibody against CD22, the antibody against CD19, the antibody against CXCR5, and the antibody against CD49d are each conjugated to a different fluorochrome having a distinguishably distinct fluorescence emission; 
 (iv) CD20, CD45, CD62L, CD39, major histocompatibility complex, class II, DR (HLADR), CD19, CD27, and CD31, wherein the antibody against CD20, the antibody against CD45, the antibody against CD62L, the antibody against CD39, the antibody against HLADR, the antibody against CD19, the antibody against CD27, and the antibody against CD31 are each conjugated to a different fluorochrome having a distinguishably distinct fluorescence emission; 
 (v) CD4, CD45, CD7, CD26, CD3, CD2, CD28, and CD8, wherein the antibody against CD4, the antibody against CD45, the antibody against CD7, the antibody against CD26, the antibody against CD3, the antibody against CD2, the antibody against CD28, and the antibody against CD8 are each conjugated to a different fluorochrome having a distinguishably distinct fluorescence emission; 
 (vi) CD4, CD45, CD27, CCR7, CD3, CD45RO, CD45RA, and CD8, wherein the antibody against CD4, the antibody against CD45, the antibody against CD27, the antibody against CCR7, the antibody against CD3, the antibody against CD45RO, the antibody against CD45RA, and the antibody against CD8 are each conjugated to a different fluorochrome having a distinguishably distinct fluorescence emission; 
 (vii) CD4, CD45, CD5, CD25, CD3, HLADR, T cell leukemia/lymphoma 1 (cyTCL1), and CD8, wherein the antibody against CD4, the antibody against CD45, the antibody against CD5, the antibody against CD25, the antibody against CD3, the antibody against HLADR, the antibody against cyTCL1, and the antibody against CD8 are each conjugated to a different fluorochrome having a distinguishably distinct fluorescence emission; 
 (viii) CD4, CD45, CD57, CD30, CD3, CD11c, and CD8, wherein the antibody against CD4, the antibody against CD45, the antibody against CD57, the antibody against CD30, the antibody against CD3, the antibody against CD11c, and the antibody against CD8 are each conjugated to a different fluorochrome having a distinguishably distinct fluorescence emission; 
 (ix) CD4, CD45, cyPerforin, cyGranzyme, CD3, CD16, CD94, and CD8, wherein the antibody against CD4, the antibody against CD45, the antibody against cyPerforin, the antibody against cyGranzyme, the antibody against CD3, the antibody against CD16, the antibody against CD94, and the antibody against CD8 are each conjugated to a different fluorochrome having a distinguishably distinct fluorescence emission; 
 (x) CD4, CD45, CD279, CD3, and CD8, the antibody against CD4, the antibody against CD45, the antibody against CD279, the antibody against CD3, and the antibody against CD8 are each conjugated to a different fluorochrome having a distinguishably distinct fluorescence emission; 
 (xi) CD2, CD45, CD7, CD26, CD3, CD56, CD5, and CD19, the antibody against CD2, the antibody against CD45, the antibody against CD7, the antibody against CD26, the antibody against CD3, the antibody against CD56, the antibody against CD5, and the antibody against CD19 are each conjugated to a different fluorochrome having a distinguishably distinct fluorescence emission; 
 (xii) CD16, CD45, CD57, CD25, CD3, CD56, CD11c, and CD19, the antibody against CD16, the antibody against CD45, the antibody against CD57, the antibody against CD25, the antibody against CD3, the antibody against CD56, the antibody against CD11c, and the antibody against CD19 are each conjugated to a different fluorochrome having a distinguishably distinct fluorescence emission; 
 (xiii) HLADR, CD45, cyPerforin, cyGranzyme, CD3, CD56, CD94, and CD19, wherein the antibody against HLADR, the antibody against CD45, the antibody against cyPerforin, the antibody against cyGranzyme, the antibody against CD3, the antibody against CD56, the antibody against CD94, and the antibody against CD19 are each conjugated to a different fluorochrome having a distinguishably distinct fluorescence emission; or 
 (xiv) CD45, CD138, CD38, CD28, CD27, CD19, CD117, and CD81, the antibody against CD45, the antibody against CD138, the antibody against CD38, the antibody against CD28, the antibody against CD27, the antibody against CD19, the antibody against CD117, and the antibody against CD81 are each conjugated to a different fluorochrome having a distinguishably distinct fluorescence emission. 
 
     
     
       3. The reagent composition or set of reagent compositions according to  claim 1  or  2 , wherein each reagent composition comprises antibodies conjugated to pacific blue (PacB) or Horizon V450, pacific orange (PacO) or aminomethylcoumarin (AMCA), fluorescein isothiocyanate (FITC) or Alexa488, phycoerythrin (PE), peridinin chlorophyll protein/cyanine 5.5 (PerCP-Cy5.5), peridinin chlorophyll protein (PerCP) or PE-TexasRed, phycoerythrin/cyanine7 (PE-Cy7), allophycocyanine (APC) or Alexa647, and allophycocyanine/H7 (APC-H7) allophycocyanine/H7 (APC-Cy7), Alexa680 or Alexa700. 
     
     
       4. A diagnostic kit for flow cytometric immunophenotyping of leukocytes comprising the set of at least two reagent compositions according to  claim 2 , and instructions for use, buffer, and/or control samples. 
     
     
       5. The diagnostic kit according to  claim 4  for the identification and characterization of mature lymphoid cells, comprising at least two further reagent compositions recited in  claim 2  under (i) through (xiv). 
     
     
       6. A method for flow cytometric immunophenotyping of leukocytes and characterization of normal and aberrant subpopulations thereof, comprising the steps of
 (a) providing a biological sample comprising leukocytes; 
 (b) contacting a first aliquot of said sample with a first reagent composition of the set according to  claim 2  and contacting at least a second aliquot of said sample with a further reagent composition of said set; 
 (c) analyzing leukocytes in said aliquots in a flow cytometer; and 
 (d) storing and evaluating the flow cytometric data obtained to characterize the leukocytes for the presence of normal and aberrant subpopulations. 
 
     
     
       7. The method according to  claim 6 , wherein said sample is peripheral blood, bone marrow, tissue sample, or a body fluid. 
     
     
       8. The method according to  claim 6 , wherein step (c) comprises the use of software for data integration and multidimensional analysis of flow cytometry files. 
     
     
       9. The method according to  claim 8 , wherein the software is INFINICYT™.